Divergen properties of two human lymphocytic cell lines isolated from a single specimen of peripheral blood

Summary Two lymphocytic cell lines were obtained from a single specimen of peripheral blood buffy coat from a patient with acute lymphoblastic leukemia who concurrently developed classical infectious mononucleosis. These two cell lines exhibit distinguishing differences in culture in parallel with reviously described differences in the cells when implanted into eonatal Syrian hamsters. These observations suggest that the cell lines were derived from two pre-existing different classes or “stelines” of cells—one of which resembles other cell lines derived from patients with acute lymphoblastic leukemia, whereas the other resembles cell lines derived from other, patients with infecttious mononucleosis. These studies were suported in part by research Grants C-6516 from the National Cancer Institute and FR-05526 from the Division of Research Facilities and Resources, National Institutes of Health.     

Differences in thermosensitization among cloned cell lines isolated from a single human melanoma xenograft

Differences in thermosensitization (effect of step-down heating) among one uncloned and five cloned cell lines isolated from a single tumor of a human melanoma xenograft were studied. Cells from passages 7-12 in vitro were exposed to graded heat treatments at 41.5 degrees C immediately, 1 h, and 2 h after a conditioning treatment of 43.5 degrees C (90 min). The thermosensitization was largest immediately after the conditioning treatment and then decayed exponentially. The differences among the cell lines were reflected in the maximum magnitude as well as in the rate of decay of the thermosensitization. The maximum thermosensitization ratios (TSR), calculated as the ratio of the D0 values at 41.5 degrees C for single-heated and preheated cells, ranged from 5.3 +/- 1.5 to 14.9 +/- 5.2 and were not correlated to the surviving fractions after the conditioning treatment. The half-times for the decay of the thermosensitization ranged from 1.5 +/- 0.3 h to 3.1 +/- 0.5 h and were not correlated to the maximum TSR. Moreover, there was no correlation between the magnitude of the maximum thermosensitization at 41.5 degrees C and the magnitude of the maximum thermotolerance at 43.5 degrees C, as induced by the same treatment (43.5 degrees C for 90 min).     

Cytostatic and cytolytic effects of human recombinant tumor necrosis factor on human renal cell carcinoma cell lines derived from a single surgical specimen

The purpose of this study was to determine whether human tumor cell lines derived from a single tumor exhibit heterogeneous responses to the anti-tumor effects of human recombinant tumor necrosis factor (h-r-TNF). Several cell lines with different metastatic propensities have been established in culture from a single surgical specimen of a human renal cell carcinoma following different selection procedures in nude mice. The cell lines exhibited significant differences in in vitro susceptibilities to cytotoxic effects of TNF. Kinetic analysis of the interaction of TNF with susceptible renal carcinoma cells demonstrated that a short 30 min interaction of TNF with the cells is sufficient to produce significant lysis 72 hr later. One cycle of exposure of sensitive cells to h-r-TNF did not produce cells resistant to it, nor did the degree of sensitivity to h-r-TNF vary after one passage of the cells in nude mice. Tumor cell resistance to the effects of TNF did develop spontaneously after prolonged cultivation in culture. We conclude that tumor cells derived from a single human renal cell carcinoma exhibit a heterogeneous response to the cytotoxic effects of h-r-TNF.     

The establishment and characterization of two new cell lines derived from a single human colonic adenocarcinoma

Two cell lines with different in vitro growth characteristics were established from a single mucinous colonic adenocarcinoma. Epithelial cells of the line 5583-E demonstrated anchorage-dependent growth while those of line 5583-S were anchorage-independent and grew as multicellular floating spheroids. Both cell lines shared common characteristics with respect to the expression of differentiation markers (secretory component, carcinoembryonic antigen), mucins and karyotype (trisomy 12 and 14, marker chromosome) but also showed consistent differences. In nude mice 5583-S cells formed moderately differentiated mucinous adenocarcinomas with high carcinoembryonic antigen and mucin production, whereas 5583-E xenografts were poorly differentiated and almost entirely failed to produce carcinoembryonic antigen and mucins. The plating efficiency of 5583-E cells appeared to be greater and doubling time shorter than those of 5583-S cells. Furthermore, 5583-E cells showed an extra isochromosome, 1q. The cell lines were genotypically and phenotypically stable over a period of 2 years. Our results reemphasize that multiple cell lines with heterogeneous phenotypic and genotypic characteristics can be obtained from a single primary tumor.     

Novel peripheral blood-derived human cell lines with properties of megakaryocytes

For 18 mo, we derived 18 cell lines from 11 donors with various clinical profiles ranging from normal to leukemic. Suspension cultures were initiated with 1 X 10(6) mononuclear blood cells/ml of nutrient medium containing 10% human serum and 10% lectin-stimulated human lymphocyte conditioned medium. The cultures were monitored weekly by morphological analyses of Wright-Giemsa-stained cell preparations. All successful cultures showed a significant decline in viability during the first 3-4 wk with rate "lymphoid" cells observed in mitosis. Within the next 2 wk, the proliferating cells gave rise to a rapidly expanding population of mononuclear cells. As the cultures expanded, cell morphology became heterogeneous with respect to cell size and nuclear ploidy, with an accumulation of giant multinuclear cells that were suggestive of megakarocytes. Even though the cells did not have the classical morphology of mature platelet-forming megakaryocytes, 90% of the cells within a cell line were positive by direct or indirect immunofluorescence for the platelet membrane glycoproteins IIb and IIIa; for surface markers HLA-Dr and B2-microglobulin; for intracellular platelet-derived growth factor and platelet factor IV; and for membrane affinity or binding with serum platelet-derived growth factor and platelet factor IV. These results suggest that a blood precursor cell, most likely a primitive megakaryoblast, was isolated from the peripheral blood and was provided with an optimal culture environment for sustained growth. These cells did not mature to a more differentiated stage, perhaps owing to regulatory factor deficiencies in this in vitro system. The remarkable frequency of obtaining cell lines with megakaryocyte properties from normal peripheral blood and the capacity of some normal donors to repeatedly yield these cell lines make this cell culture system indeed unique by being selective for putative megakaryocyte precursors.     

Comprehensive DNA methylation analysis of human peripheral blood leukocytes and lymphoblastoid cell lines

DNA methylation is involved in development and in human diseases. Genomic DNA derived from lymphoblastoid cell lines (LCLs) is commonly used to study DNA methylation. There are potential confounding factors regarding the use of LCL-derived DNA, however, such as Epstein-Barr (EB) viral infection and artifacts induced during cell culture. Recently, several groups compared the DNA methylation status of peripheral blood leukocytes (PBLs) and LCLs and concluded that the DNA methylation profiles between them might be consistent. To confirm and extend theses results, we performed a comprehensive DNA methylation analysis using both PBLs and LCLs derived from the same individuals. Using the luminometric methylation assay, we revealed that the global DNA methylation level was different between PBLs and LCLs. Furthermore, the direction of change was not consistent. Comparisons of genome-wide DNA methylation patterns of promoter regions revealed that methylation profiles were largely conserved between PBLs and LCLs. A preliminary analysis in a small number of samples suggested that the methylation status of an LCL may be better correlated with PBLs from the same individual than with LCLs from other individuals. Expectedly, DNA methylation in promoter regions overlapping with CpG islands was associated with gene silencing in both PBLs and LCLs. With regard to methylation differences, we found that hypermethylation was more predominant than hypomethylation in LCLs compared with PBLs. These findings suggest that LCLs should be used for DNA methylation studies with caution as the methylation patterns of promoter regions in LCLs are not always the same as those in PBLs.Key words: DNA methylation, lymphoblastoid cell lines, peripheral blood leukocytes, LUMA, promoter tiling array, gene expression     

Comprehensive DNA methylation analysis of human peripheral blood leukocytes and lymphoblastoid cell lines

DNA methylation is involved in development and in human diseases. Genomic DNA derived from lymphoblastoid cell lines (LCLs) is commonly used to study DNA methylation. There are potential confounding factors regarding the use of LCL-derived DNA, however, such as Epstein-Barr (EB) viral infection and artifacts induced during cell culture. Recently, several groups compared the DNA methylation status of peripheral blood leukocytes (PBLs) and LCLs and concluded that the DNA methylation profiles between them might be consistent. To confirm and extend theses results, we performed a comprehensive DNA methylation analysis using both PBLs and LCLs derived from the same individuals. Using the luminometric methylation assay, we revealed that the global DNA methylation level was different between PBLs and LCLs. Furthermore, the direction of change was not consistent. Comparisons of genome-wide DNA methylation patterns of promoter regions revealed that methylation profiles were largely conserved between PBLs and LCLs. A preliminary analysis in a small number of samples suggested that the methylation status of an LCL may be better correlated with PBLs from the same individual than with LCLs from other individuals. Expectedly, DNA methylation in promoter regions overlapping with CpG islands was associated with gene silencing in both PBLs and LCLs. With regard to methylation differences, we found that hypermethylation was more predominant than hypomethylation in LCLs compared with PBLs. These findings suggest that LCLs should be used for DNA methylation studies with caution as the methylation patterns of promoter regions in LCLs are not always the same as those in PBLs.     

Characterization of two types of osteoclasts from human peripheral blood monocytes

The two osteoclastogenesis pathways, receptor activator nuclear factor (NF)-kappaB ligand (RANKL)-mediated and fusion regulatory protein-1 (FRP-1)-mediated osteoclastogenesis, have recently been reported. There were significant differences in differentiation and activation mechanisms between the two pathways. When monocytes were cultured with FRP-1 without adding M-CSF, essential for the RANKL system, TRAP-positive polykaryocyte formation occurred. FRP-1-mediated osteoclasts formed larger pits on mineralized calcium phosphate plates than RANKL+M-CSF-mediated osteoclasts did. Lacunae on dentin surfaces induced by FRP-1-mediated osteoclasts were inclined to be single and isolated. However, osteoclasts induced by RANKL+M-CSF made many connected pits on dentin surfaces as if they crawled on there. Interestingly, FRP-1 osteoclastogenesis was enhanced by M-CSF/IL-1alpha, while chemotactic behavior to the dentin slices was not effected. There were differences in pH and concentration of HCO3- at culture endpoint and in adherent feature to dentin surfaces. Our findings indicate there are two types of osteoclasts with distinct properties.     

Monitoring repair of DNA damage in cell lines and human peripheral blood mononuclear cells

We introduce a method to follow DNA repair that is suitable for both clinical and laboratory samples. An episomal construct with a unique 8-oxoguanine (8-oxoG) base at a defined position was prepared in vitro using single-stranded phage harboring a 678-bp tract from exons 5 to 9 of the human P53 gene. Mixing curve experiments showed that the real-time PCR method has a linear response to damage, suggesting that it is useful for DNA repair studies. The episomal construct with a unique 8-oxoG base was introduced into AD293 cells or human peripheral blood mononuclear cells, and plasmids were recovered as a function of time. The quantitative real-time PCR assay demonstrated that repair of the 8-oxoG was 80% complete in less than 48 h in AD293 cells. Transfection of small interfering RNAs down-regulated OGG1 expression in AD293 cells and reduced the repair of 8-oxoG to 30%. Transfection of the episome into unstimulated white blood cells showed that 8-oxoG repair had a half-life of 2 to 5 h. This method is a rapid, reproducible, and robust way to monitor repair of specific adducts in virtually any cell type.     

Biological properties of two newly established cell lines (RMUG-S, RMUG-L) from a human ovarian mucinous cystadenocarcinoma

Two morphological different cell lines (RMUG-S and RMUG-L) have been established from a human ovarian mucinous cystadenocarcinoma. CA-125 and CEA were demonstrated in the patient's serum. PAS, Alcian blue and mucicarmine positive substances were observed in the original tumor and two cultured cells. The modal chromosomal numbers of RMUG-S and RMUG-L were stable in the hypodiploid and hyper-triploid ranges, respectively. Radioimmunoassay and immunocytochemical staining revealed that RMUG-S and RMUG-L mainly produced CA-125 and CEA, respectively. In CBA nude mouse, RMUG-S and RMUG-L cells mainly produced poorly-differentiated adenocarcinoma, but in mucinous cystadenocarcinoma was detected in part of the RMUG-L tumor.     

Differential spontaneous killing of human and murine tumour cell lines by leucocyte subpopulations from carp peripheral blood leucocytes

Cell populations from carp (Cyprinus carpio L.) peripheral blood leucocytes (PBLs) were examined for nonspecific cytotoxicities. By using monoclonal antibodies (MAbs) against carp thrombocytes (TCL-HB8) and both neutrophils and monocytes (TCL-BE8), PBLs with a density of 1.08 g ml-1 were separated into three fractions: thrombocytes, a mixture of neutrophils and monocytes, and other cells (mainly lymphocytes), and the separated cells were tested for cytotoxic activities against mammalian tumour cell lines (K562, HeLa, P815 and Yac-1 cell). Consequently, the mixture of neutrophils and monocytes exhibited cytolysis against these target cells, whereas the lymphocyte-rich and thrombocyte fractions did not show any cytolysis. To isolate only neutrophils, which do not contain monocytes, the MAb (TCL-BE8) positive cells from PBLs with a density of 1.08-1.09 g ml-1 were separated. Pure isolated neutrophils showed cytotoxic activities against K562 cells, but not P815 cells. Furthermore, analysis of the cytolytic mechanisms indicated that killing of these cells depended on H2O2 or HOCl. These results suggest that both neutrophils and monocytes are effectors for nonspecific cytotoxicity in carp PBLs, and neutrophils may be distinct from monocytes in their reactivity in cytolysis, including target cell selectivity and/or target cell sensitivity, and the cytolytic pathway. In carp, cytotoxicity of target cells can be mediated by several populations of their leucocytes which have cytotoxic capacities with various recognition and cytolytic mechanisms.     

Characterization of plasminogen activator from two human renal carcinoma cell lines

Plasminogen activator (PA) activity was identified in the conditioned medium of two human renal carcinoma cell lines, Cur and Caki-1. PA activity of medium, following chromatography on Con A-Sepharose, was divided into effluent and eluate fractions, the latter obtained after elution with methyl mannoside. The ratio of PA activity in effluent:eluate was 90:10 for Caki-1 and 60:40 for Cur. The PA of both effluent fractions and the Caki-1 eluate fraction was of the urokinase (UK) type. Identification rested on molecular weight determination by zymography (major component with Mr 52,000 and a less prominent component of 93,000), lack of binding to fibrin, inhibition by anti-UK antibodies, and lack of inhibitory effect of anti-tissue type PA (TPA) antibodies or the Erythrina trypsin inhibitor, which inhibits TPA but not UK. PA of the Cur eluate fraction gave a more complex pattern in that it bound significantly to fibrin (like TPA), was completely inhibited by both anti-UK and anti-TPA antibodies, but was unaffected by Erythrina trypsin inhibitor. These results raise the possibility of an unusual PA-like enzyme that immunologically cross reacts with anti-UK and anti-TPA. Most of the PA of both cell lines was secreted in a latent form that could be activated by trypsin treatment. The latency appears to result largely from secretion of urokinase proenzyme, which is consistent with the Mr 52,000 of the major PA species and the insensitivity to diisopropyl fluorophosphate inhibition prior to trypsin activation. However, in addition, a UK binding component was found in the conditioned medium, which produced an Mr 93,000 component by reaction with UK.     

Opposing effects of human peripheral blood lymphocytes on the growth of cultured leukemia cell lines

Natural killing of two human leukemia cell lines (K562 and Molt 4) in a soft-agar, clonagenic assay was shown to be the result of two opposite yet concurrent processes: target cell colony stimulation and inhibition. The stimulatory effect was demonstrable when the effector lymphocytes and target cells were separated in contiguous agar layers, suggesting mediation by a soluble factor. Similarly, stimulation occurred when the effector lymphocytes and target cells were combined at low effector-target cell ratios that do not favor direct cell contact. Target colony inhibition was found to be dominant when large E:T ratios were employed. Both target-effector binding and natural killing were significantly reduced in medium devoid of divalent cations.     

Divergent human populations show extensive shared IGK rearrangements in peripheral blood B cells

We have analysed the transcribed immunoglobulin kappa (IGK) repertoire of peripheral blood B cells from four individuals from two genetically distinct populations, Papua New Guinean and Australian, using high-throughput DNA sequencing. The depth of sequencing data for each individual averaged 5,548 high-quality IGK reads, and permitted genotyping of the inferred IGKV and IGKJ germline gene segments for each individual. All individuals were homozygous at each IGKJ locus and had highly similar inferred IGKV genotypes. Preferential gene usage was seen at both the IGKV and IGKJ loci, but only IGKV segment usage varied significantly between individuals. Despite the differences in IGKV gene utilisation, the rearranged IGK repertoires showed extensive identity at the amino acid level. Public rearrangements (those shared by two or more individuals) made up 60.2% of the total sequenced IGK rearrangements. The total diversity of IGK rearrangements of each individual was estimated to range from just 340 to 549 unique amino acid sequences. Thus, the repertoire of unique expressed IGK rearrangements is dramatically less than previous theoretical estimates of IGK diversity, and the majority of expressed IGK rearrangements are likely to be extensively shared in individual human beings.     

Characterization of cancer cell lines established from two human metastatic breast cancers

Summary Cell lines are valuable resources for the study of the malignancy and potential therapy of human breast cancer. A major problem with adapting fresh breast tumor specimens to grow in vitro is contamination by fibroblasts. Previously, we have reported a technique to overcome this problem (Nayak, S. K; Dillman, R. O. Clin. Biotechnol. 3:237–242; 1991). We have recently established two new breast cancer cell lines, HH315 and HH375, that were derived from abdominal and supraclavicular lymph node metastases from two patients. They were characterized by (1) growth kinetics; (2) staining with monoclonal antibodies (MoAbs) to cytokeratin-19, epithelial membrane antigen (EMA), anticarcinoembryonic antigen (CEA), breast cancer antigen 1 (BRST-1), breast cancer antigen 2 (BRST-2), Her2/neu, and p53; (3) expression of domains of urinary plasminogen activator (uPA), neural cell adhesion molecule (NCAM), and haptoglobin (Hp) (Harvey et al., 1997); and (4) karyotypic analysis. Growth kinetic studies showed that doubling times for both lines ranged from 48 to 96 h. These two cell lines were found to have characteristics of the metastatic breast cancer cells. Both lines stained positive with MoAbs to cytokeratin-19 and EMA, thus confirming their epithelial origin. They also strongly reacted with the pan-breast carcinoma MoAbs BRST-1 and BRST-2, and carcinoembryonic CEA MoAb. Both cell lines overexpressed the oncogene proteins Her2/neu and p53. The tumor cells were negative for estrogen and progesterone receptors. HH315 cells were poorly differentiated, whereas the HH375 cells exhibited adenocarcinoma morphology. Both cell lines showed intense cell surface and some cytoplasmic staining for uPA, NCAM, and Hp domains, which is a characteristic of malignant neoplasms (Harvey et al., 1997). The HH375 cell line showed two cell types, of which 60% were hyperdiploids with 60–70 chromosomes and 5–10 marker chromosomes. The remaining cells were polyploid with more than 200 chromosomes. Cell line HH315 consisted of only a polyploid population. These cell lines may be useful in breast cancer research.     

Heterogeneity in radiosensitization by heat of cloned cell lines derived from a single human melanoma xenograft

Heterogeneity in radiosensitization by heat was studied using one uncloned and five cloned cell lines isolated from a single tumour of a human melanoma xenograft. Cells from passages 7-12 in vitro were given heat treatments of 42.5 degrees C (45 min), 43.5 degrees C (45 min) or 44.5 degrees C (45 min) immediately after exposure to graded doses of radiation. The survival curves after irradiation alone had similar D0 values but differed in the size of the shoulder. The heterogeneity in heat radiosensitization was reflected in differences in decrease of the D0 values. The thermal enhancement ratios, calculated from the D0 values, were in the ranges 1.2 +/- 0.2-1.7 +/- 0.2 (42.5 degrees C), 1.4 +/- 0.3-2.4 +/- 0.4 (43.5 degrees C) and 2.3 +/- 0.4-3.4 +/- 0.4 (44.5 degrees C). Moreover, at 43.5 degrees C the heterogeneity was also reflected in different modifications of the shape of the survival curves. Two lines showed survival curves with a significant shoulder and a relatively low D0 value whereas two other lines had lost the shoulder almost completely but showed relatively high D0 values. All lines showed survival curves with a broad shoulder after heating at 42.5 degrees C, whereas none of the lines showed survival curves with a significant shoulder after heating at 44.5 degrees C.